Journal: The EMBO Journal

Article Title: eIF3 mRNA selectivity profiling reveals eIF3k as a cancer‐relevant regulator of ribosome content

doi: 10.15252/embj.2022112362

Figure Lengend Snippet: Lysates of eIF3k‐mAID cells exposed to DMSO or IAA for 12 h were separated by sucrose density gradient centrifugation. Total ribosome content was determined by integrating the monosomal and polysomal peaks and plotted. Error bars represent means ± SD, n = 3. Numbers indicate P ‐values (unpaired Student's t ‐test). 18S and 28S rRNA levels were determined by RT–qPCR across a sucrose density gradient. Bars represent mean rRNA levels in summed monosomal and polysomal fractions ± SD, n = 3; numbers indicate P ‐values (unpaired Student's t ‐test). Total ribosome occupancy of the indicated mRNAs in eIF3k‐mAID cells exposed to DMSO or IAA for 12 h was determined by RT–qPCR of RNA across a sucrose density gradient (see ). Bars represent means ± SD, n = 3; numbers indicate P ‐values (unpaired Student's t ‐test). Triplicate RT–qPCR data across the sucrose gradient are shown below the bar graphs. Equal numbers of eIF3k‐mAID cells stably expressing ectopic RPS15A (pCDH‐RPS15A) or empty vector (pCDH) were plated and counted over a period of 6 days. Graphs represent means ± SD, n = 3. Numbers indicate P ‐values (two‐stage step‐up method of Benjamini, Krieger, and Yekutieli). 1 × 10 6 eIF3k‐mAID cells stably expressing ectopic RPS15A (pCDH‐RPS15A) or empty vector (pCDH) were injected into nude mice and tumor growth was followed for 2 weeks. Graphs represent means ± SD, n = 5–7 (see Fig ). Numbers indicate P ‐values (two‐stage step‐up method of Benjamini, Krieger, and Yekutieli). Lysate of eIF3k‐mAID cells stably expressing ectopic RPS15A (pCDH‐RPS15A) or empty vector (pCDH) were separated by sucrose density gradient centrifugation. Total ribosome content was determined by integrating and summing the monosomal and polysomal peak areas. Error bars represent means ± SD, n = 3. Numbers indicate P ‐values (unpaired Student's t ‐test). Equal numbers of eIF3k‐mAID cells stably expressing ectopic RPS4X (pCDH‐RPS4X) or empty vector (pCDH) were plated and counted over a period of 6 days. Graphs represent means ± SD, n = 3. Numbers indicate P ‐values (two‐stage step‐up method of Benjamini, Krieger, and Yekutieli). Total ribosome content of these cells was determined as described in (A). Steady‐state free ribosomal pool and translation rate as a function of eIF3k concentration (top two graphs) and the steady‐state translation rate as a function of eIF3a concentration (bottom graph). The concentration of other eIF3 subunits is kept constant at 0.5. Data information: n = number of biological replicates. Source data are available online for this figure.

Article Snippet: RPS15A sgRNA‐2 lentiCRISPR v2 plasmid , This manuscript , Addgene submission pending.

Techniques: Gradient Centrifugation, Quantitative RT-PCR, Stable Transfection, Expressing, Plasmid Preparation, Injection, Concentration Assay

Journal: The EMBO Journal

Article Title: eIF3 mRNA selectivity profiling reveals eIF3k as a cancer‐relevant regulator of ribosome content

doi: 10.15252/embj.2022112362

Figure Lengend Snippet: Total ribosome occupancy of the indicated mRNAs in eIF3k‐mAID cells exposed to DMSO or IAA for 48 h was determined by RT–qPCR of RNA across a sucrose density gradient (see ). Bars represent means ± SD, n = 3; numbers indicate P ‐values (unpaired Student's t ‐test). Triplicate RT–qPCR data across the sucrose gradient are shown below the bar graphs. 1 × 10 6 eIF3k‐mAID cells stably expressing ectopic RPS15A (pCDH‐RPS15A) or empty vector (pCDH) were injected into nude mice and tumor growth was followed for 2 weeks. Graphs represent means of final tumor weights ± SD, n = 6. Numbers indicate P ‐values (unpaired Student's t ‐test). Simulated effect of eIF3k and eIF3a concentration on the average steady‐state density and traffic jam. The concentration of other eIF3 subunits is kept constant at 0.5. Graph depicting the free ribosome pool k‐gain as a function of the eIF3k concentration. See for details. Data information: n = number of biological replicates. Source data are available online for this figure.

Article Snippet: RPS15A sgRNA‐2 lentiCRISPR v2 plasmid , This manuscript , Addgene submission pending.

Techniques: Quantitative RT-PCR, Stable Transfection, Expressing, Plasmid Preparation, Injection, Concentration Assay

Journal: The EMBO Journal

Article Title: eIF3 mRNA selectivity profiling reveals eIF3k as a cancer‐relevant regulator of ribosome content

doi: 10.15252/embj.2022112362

Figure Lengend Snippet: 5′‐UTR sequences of RPS15A and RPS4X. 5′‐TOP element known to boost translation of ribosomal protein mRNAs (Meyuhas, ) are highlighted in gold prints, the eIF3‐binding sites mapped by Lee et al and Meyer et al are highlighted in red print. The alleles created by gene editing are shown (S15A‐eIF3KO, S4X‐eIF3KO). Note that the eIF3‐binding site in RPS4X is upstream of or overlapping with the major transcription start site. Thus, the S4X‐eIF3KO truncation we failed to generate most likely abolished the transcription of RPS4X mRNA. Parental eIF3k‐mAID cells or S15A‐eIF3KO cells (clones #45 and #361) were maintained in standard media, and cell numbers were determined at various time points. Data represent means ± SD, n = 3. Asterisks denote: * P < 0.005, ** P < 0.00005, *** P < 0.000005 (two‐stage step‐up method of Benjamini, Krieger, and Yekutieli). Relative levels of mRNAs encoding RPS15A , RPS4X , and RPL7A before and after depletion of eIF3k were determined in the indicated cell lines by RT–qPCR. Data were normalized to the signal obtained for GAPDH. Bars represent means ± SD, n = 3; numbers indicate P ‐values (unpaired Student's t ‐test). Relative translational efficiencies (TEs) of mRNAs encoding RPS15A , RPS4X , and RPL7A before and after depletion of eIF3k were determined in the indicated cell lines. RT–qPCR was performed on total RNA and on RNA contained within polysomal fractions > 2 ribosomes, and TE was calculated according to the formula TE = polysomal mRNA / total mRNA. Bars represent means ± SD, n = 3; numbers indicate P ‐values (unpaired Student's t ‐test). Basal expression of the indicated proteins was determined in parental eIF3k‐mAID cells or S15A‐eIF3KO cells (clones #45 and #361) by immunoblotting, followed by quantification of the blots. Bars represent means ± SD, n = 3 (Fig ). Numbers indicate P ‐values (unpaired Student's t ‐test). Data information: n = number of biological replicates. Source data are available online for this figure.

Article Snippet: RPS15A sgRNA‐2 lentiCRISPR v2 plasmid , This manuscript , Addgene submission pending.

Techniques: Binding Assay, Clone Assay, Quantitative RT-PCR, Expressing, Western Blot

Journal: The EMBO Journal

Article Title: eIF3 mRNA selectivity profiling reveals eIF3k as a cancer‐relevant regulator of ribosome content

doi: 10.15252/embj.2022112362

Figure Lengend Snippet: Parental eIF3k‐mAID cells or S15A‐eIF3KO cells (clones #45 and #361) were maintained in media with DMSO or IAA, and cell numbers were determined at various time points. Data represent means ± SD (too small to be visible), n = 3. All P ‐values (two‐stage step‐up method of Benjamini, Krieger, and Yekutieli) were > 0.45 except at the single time point where indicated otherwise. 1 × 10 6 S15A‐eIF3KO (clone #361) cells were injected into nude mice. When tumors reached a diameter of ~ 4 mm, mice were treated with vehicle or 500 mg/kg IAA, and tumor growth was measured for 2 weeks. Graphs represent means ± SD, n = 5–7. Numbers indicate P ‐values (two‐stage step‐up method of Benjamini, Krieger, and Yekutieli). Lysate of S15A‐eIF3KO cells (clones #45 and #361) were separated by sucrose density gradient centrifugation. Total ribosome content was determined by integrating and summing the monosomal and polysomal peak areas. Error bars represent means ± SD, n = 3 (see Fig ). Numbers indicate P ‐values (unpaired Student's t ‐test). Total ribosome occupancy of the indicated mRNAs in S15A‐eIF3KO (clone #361) cells exposed to DMSO or IAA for 12 h was determined by RT–qPCR of RNA across a sucrose density gradient (see ). Bars represent means ± SD, n = 3; numbers indicate P ‐values (unpaired Student's t ‐test). Triplicate RT–qPCR data across the sucrose gradient are shown below the bar graphs. RNA immunoprecipitation. eIF3k‐mAID and S15A‐eIF3KO (clone #361) cells were exposed to DMSO or IAA for 12 h. Cell lysates were employed in immunoprecipitation with eIF3c antibodies and co‐precipitated mRNAs were quantified by qPCR. Bars represent means ± SD, n = 4; numbers indicate P ‐values (unpaired Student's t ‐test). Equal numbers of S15A‐eIF3KO (clone #361) cells stably expressing ectopic RPS15A (pCDH‐RPS15A) or empty vector (pCDH) were plated and counted over a period of 6 days. Graphs represent means ± SD, n = 3. Asterisks denote: * P < 0.005, ** P < 0.00005, *** P < 0.000005 (two‐stage step‐up method of Benjamini, Krieger, and Yekutieli). Data information: n = number of biological replicates. Source data are available online for this figure.

Article Snippet: RPS15A sgRNA‐2 lentiCRISPR v2 plasmid , This manuscript , Addgene submission pending.

Techniques: Clone Assay, Injection, Gradient Centrifugation, Quantitative RT-PCR, RNA Immunoprecipitation, Immunoprecipitation, Stable Transfection, Expressing, Plasmid Preparation

Journal: The EMBO Journal

Article Title: eIF3 mRNA selectivity profiling reveals eIF3k as a cancer‐relevant regulator of ribosome content

doi: 10.15252/embj.2022112362

Figure Lengend Snippet: When the eIF3‐binding site in the 5′‐UTR is occupied by holo‐eIF3 containing the eIF3k‐l module, RPS15A mRNA is translated efficiently at basal rate. When the eIF3k‐l module is missing (or functionally inactivated, perhaps degraded in response to stress conditions), the remaining eIF3 complex boosts maximal translation of RPS15A mRNA through the eIF3‐binding site. The extra RPS15A then boosts ribosome content, translation, and growth, for example by completing substoichiometric ribosomes.

Article Snippet: RPS15A sgRNA‐2 lentiCRISPR v2 plasmid , This manuscript , Addgene submission pending.

Techniques: Binding Assay

Journal: The EMBO Journal

Article Title: eIF3 mRNA selectivity profiling reveals eIF3k as a cancer‐relevant regulator of ribosome content

doi: 10.15252/embj.2022112362

Figure Lengend Snippet:

Article Snippet: RPS15A sgRNA‐2 lentiCRISPR v2 plasmid , This manuscript , Addgene submission pending.

Techniques: Recombinant, Plasmid Preparation, Sequencing, Protease Inhibitor, Magnetic Beads, Software, Microscopy, Microarray, Imaging, Flow Cytometry, Mutagenesis, Gel Extraction, cDNA Synthesis, Sample Prep, Clear Native PAGE, Bicinchoninic Acid Protein Assay

Journal: Clinical and Translational Medicine

Article Title: Genome‐wide profiling of N6‐methyladenosine‐modified pseudogene‐derived long noncoding RNAs reveals the tumour‐promoting and innate immune‐restraining function of RPS15AP12 in ovarian cancer

doi: 10.1002/ctm2.70249

Figure Lengend Snippet: RPS15AP12‐lncRNA competitively combines miR‐96‐3p to positively regulate RPS15A expression. (A) Spearman's correlation analysis between RNA levels of RPS15AP12 and RPS15A according to the TCGA ovarian cancer cohort. (B) RT‐qPCR assays detecting the expression of RPS15A upon RPS15AP12 KO in OVCAR3 and SKOV3 cell lines. (C) Western blot detecting the protein level of RPS15A upon RPS15AP12 KO in OVCAR3 and SKOV3 cell lines. (D) Representative IHC staining images and quantitative analysis of RPS15A in xenograft tumours from RPS15AP12 knockout and control cells treated nude mice. Scale bar, 100 µm. Statistical analyses showed the IHC staining of RPS15A from xenograft tumours. (E) AGO2 RIP‐PCR detecting the binding of RPS15A and RPS15AP12 with miRNAs. (F) Venn plot showing co‐binding miRNAs shared by RPA15A and RPS15AP12. (G) Schematic diagram of the binding site and sequence in miR‐96, RPS15AP12 and RPS15A. RT‐qPCR detecting miR‐96‐3p level upon miR‐96‐3p mimics and miR‐96‐3p inhibitor in OVCAR3 and SKOV3 cell lines. (H) RT‐qPCR detecting miR‐96‐3p expression upon miR‐96‐3p mimics and miR‐96‐3p inhibitor in OVCAR3 and SKOV3 cell lines. (I) RT‐qPCR detecting RPS15A mRNA level upon miR‐96‐3p mimics and miR‐96‐3p inhibitor in OVCAR3 and SKOV3 cell lines. (J) RT‐qPCR detecting RPS15A mRNA level was performed with control, RPS15AP12‐KO, and RPS15AP12‐KO+miR‐96‐3p inhibitor in OVCAR3 and SKOV3 cell lines. RT‐qPCR detecting RPS15A mRNA level was performed with control, RPS15AP12‐OE, and RPS15AP12‐OE+miR‐96‐3p mimics in OVCAR3 and SKOV3 cell lines. (K) The schematic diagram of wild‐type and mutant 3′UTR of RPS15A for luciferase assays. (L, M) Luciferase assays of RPS15A‐WT and RPS15A‐MUT upon control, miR‐96‐3p mimics and miR‐96‐3p mimics+RPS15AP12‐OE in HEK293T and OVCAR3 cells. One‐way ANOVA, * p < .05, ** p < .01, *** p < .001; NS, not significant.

Article Snippet: Primary antibodies were as follows: METTL3 (1:1000, 15073‐1‐AP, Proteintech), FTO (1:1000, 27226‐1‐AP, Proteintech), YTHDF2 (1:2000, 24744‐1‐AP, Proteintech), RPS15A (1:1000, TA369533S, ORIGENE), MDA5 (1:2000, 21775‐1‐AP, Proteintech), RIG‐1 (1:1000, 20566‐1‐AP, Proteintech), TBK1 (1:1000, #3504, CST), p‐TBK1 (1:1000, 82383‐1‐RR, Proteintech), IRF3 (1:5000, 11312‐1‐AP, Proteintech), p‐IRF3 (1:1000, 29528‐1‐AP, Proteintech), GAPDH (1:10000, 60004‐1‐Ig, Proteintech).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Knock-Out, Control, Binding Assay, Sequencing, Mutagenesis, Luciferase

Journal: Clinical and Translational Medicine

Article Title: Genome‐wide profiling of N6‐methyladenosine‐modified pseudogene‐derived long noncoding RNAs reveals the tumour‐promoting and innate immune‐restraining function of RPS15AP12 in ovarian cancer

doi: 10.1002/ctm2.70249

Figure Lengend Snippet: RPS15AP12 inhibits the anti‐tumour effect of innate immune‐related pathways in ovarian cancer. (A) Volcano plot showing log2(fold change) and p ‐value of RNA‐seq upon RPS15A knockdown and RPS15AP12 knockout. (B) Overlapped DEGs of RNA‐seq upon RPS15A knockdown and RPS15AP12 knockout. (C) Enrichment network showing innate immune‐related pathways and corresponding hub genes by RPS15A knockdown and RPS15AP12 knockout. (D) Heatmap of 25 shared DEGs in innate immune‐related and proliferation‐related pathways in RNA‐seq upon RPS15A knockdown and RPS15AP12 knockout. (E) RT‐qPCR detecting RNA level of 25 shared DEGs in innate immune‐related and apoptosis‐related pathways in OVCAR3 and SKOV3 cell lines upon RPS15AP12 KO. (F) RT‐qPCR detecting RNA level of 25 shared DEGs in innate immune‐related pathways in OVCAR3 and SKOV3 cell lines upon RPS15A knockdown. (G) Western blot assays detecting protein expression using antibodies as indicated in OVCAR3 and SKOV3 cell lines upon RPS15A knockdown. (H) Western blot assays detecting protein expression using antibodies as indicated in OVCAR3 and SKOV3 cell lines upon RPS15AP12 depletion. One‐way ANOVA, * p < .05, ** p < .01, *** p < .001; NS, not significant.

Article Snippet: Primary antibodies were as follows: METTL3 (1:1000, 15073‐1‐AP, Proteintech), FTO (1:1000, 27226‐1‐AP, Proteintech), YTHDF2 (1:2000, 24744‐1‐AP, Proteintech), RPS15A (1:1000, TA369533S, ORIGENE), MDA5 (1:2000, 21775‐1‐AP, Proteintech), RIG‐1 (1:1000, 20566‐1‐AP, Proteintech), TBK1 (1:1000, #3504, CST), p‐TBK1 (1:1000, 82383‐1‐RR, Proteintech), IRF3 (1:5000, 11312‐1‐AP, Proteintech), p‐IRF3 (1:1000, 29528‐1‐AP, Proteintech), GAPDH (1:10000, 60004‐1‐Ig, Proteintech).

Techniques: RNA Sequencing, Knockdown, Knock-Out, Quantitative RT-PCR, Western Blot, Expressing

Journal: Cancer Medicine

Article Title: MCM8 promotes gastric cancer progression through RPS15A and predicts poor prognosis

doi: 10.1002/cam4.7424

Figure Lengend Snippet: RPS15A is the target of MCM8 in GC. (A) The differentially expressed genes (DEGs) between sh‐MCM8 and sh‐Ctrl groups. (B) Ingenuity pathway analysis (IPA) analyzed the enrichment of DEGs in the typical signal pathways. (C) The significant enrichment of DEGs in diseases and functions. (D) Network of interactions between MCM8 and classic signaling pathway genes. The expression of targets in GC cells were detected by (E) qRT‐PCR and (F) WB. (G) The binding of MCM8 and RPS15A protein was confirmed via Co‐IP assay. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: MCM8 (1:500, Proteintech, Chicago, USA), RPS15A (1:2000, Proteintech, Chicago, USA), CDK4 (1:1000, Proteintech, Chicago, USA), MAPK14 (1:2000, Proteintech, Chicago, USA), CCND1 (1:750, CST, Boston, USA), SMAD3 (1:1000, Abcam, Cambridge, UK), SMAD4 (1:500, Santa Cruz, California, USA), ARAF (1:1000, CST, Boston, USA), RPS6KA1 (1:500, Proteintech, Chicago, USA), GAPDH (1:30000, Proteintech, Chicago, USA), P53 (1:3000, Proteintech, Chicago, USA), p‐P53 (1:2000, Proteintech, Chicago, USA), STAT3 (1:1500, CST, Boston, USA), p‐STAT3 (1:500, CST, Boston, USA), c‐Jun (1:1000, Proteintech, Chicago, USA), p‐c‐Jun (1:2000, CST, Boston, USA), p70S6K (1:1000, Affinity, Cincinnati, USA), p‐p70S6K (1:1000, Affinity, Cincinnati, USA), P38α(1:2000, Abcam, Cambridge, UK), p‐P38α(1:1000, Abcam, Cambridge, UK), LYN (1:1000, CST, Boston, USA), p‐LYN (1:1000, CST, Boston, USA), PYK2 (1:2000, Abcam, Cambridge, UK), p‐PYK2 (1:1000, CST, Boston, USA), STAT1 (1:1000, Proteintech, Chicago, USA), p‐STAT1 (1:2000, Abcam, Cambridge, UK).

Techniques: Expressing, Quantitative RT-PCR, Binding Assay, Co-Immunoprecipitation Assay

Journal: Cancer Medicine

Article Title: MCM8 promotes gastric cancer progression through RPS15A and predicts poor prognosis

doi: 10.1002/cam4.7424

Figure Lengend Snippet: RPS15A knockdown inhibits the phenotypic functions of GC. (A) RPS15A expression in GC and normal tissues from TCGA. (B) Kaplan–Meier plots of GC patients with high and low expression of RPS15A. (C) RPS15A expression in GC and GES‐1 cells. (D, E) IHC staining of RPS15A in GC and normal tissues. Scale bars: 100 μm. (F, G) The proliferation ability of RPS15A was measured by (F) Celigo cell counting assay and (G) Colony formation assay. (H) Annexin V‐APC staining in sh‐RPS15A and sh‐Ctrl groups. (I–K) The migration and invasion ability of MGC‐803 cells was detected by (I) Wound‐healing assay (scale bar: 1 μm) (J, K) and Transwell assay (100×). * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: MCM8 (1:500, Proteintech, Chicago, USA), RPS15A (1:2000, Proteintech, Chicago, USA), CDK4 (1:1000, Proteintech, Chicago, USA), MAPK14 (1:2000, Proteintech, Chicago, USA), CCND1 (1:750, CST, Boston, USA), SMAD3 (1:1000, Abcam, Cambridge, UK), SMAD4 (1:500, Santa Cruz, California, USA), ARAF (1:1000, CST, Boston, USA), RPS6KA1 (1:500, Proteintech, Chicago, USA), GAPDH (1:30000, Proteintech, Chicago, USA), P53 (1:3000, Proteintech, Chicago, USA), p‐P53 (1:2000, Proteintech, Chicago, USA), STAT3 (1:1500, CST, Boston, USA), p‐STAT3 (1:500, CST, Boston, USA), c‐Jun (1:1000, Proteintech, Chicago, USA), p‐c‐Jun (1:2000, CST, Boston, USA), p70S6K (1:1000, Affinity, Cincinnati, USA), p‐p70S6K (1:1000, Affinity, Cincinnati, USA), P38α(1:2000, Abcam, Cambridge, UK), p‐P38α(1:1000, Abcam, Cambridge, UK), LYN (1:1000, CST, Boston, USA), p‐LYN (1:1000, CST, Boston, USA), PYK2 (1:2000, Abcam, Cambridge, UK), p‐PYK2 (1:1000, CST, Boston, USA), STAT1 (1:1000, Proteintech, Chicago, USA), p‐STAT1 (1:2000, Abcam, Cambridge, UK).

Techniques: Knockdown, Expressing, Immunohistochemistry, Cell Counting, Colony Assay, Staining, Migration, Wound Healing Assay, Transwell Assay

Journal: Cancer Medicine

Article Title: MCM8 promotes gastric cancer progression through RPS15A and predicts poor prognosis

doi: 10.1002/cam4.7424

Figure Lengend Snippet: Overexpression of RPS15A reverses the changes of MCM8 knockdown in GC cells. (A) The transfection efficiency was detected by WB. (B–E) sh‐Ctrl+oe‐Ctrl, sh‐Ctrl+oe‐RPS15A, sh‐MCM8+oe‐Ctrl, and sh‐MCM8+oe‐RPS15A groups were established in AGS cells. The loss or gain of function experiments showed that RPS15A reversed the changes of MCM8 knockdown in GC cells, including (B, C) proliferation, (D) apoptosis, and (E) migration (100×). * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: MCM8 (1:500, Proteintech, Chicago, USA), RPS15A (1:2000, Proteintech, Chicago, USA), CDK4 (1:1000, Proteintech, Chicago, USA), MAPK14 (1:2000, Proteintech, Chicago, USA), CCND1 (1:750, CST, Boston, USA), SMAD3 (1:1000, Abcam, Cambridge, UK), SMAD4 (1:500, Santa Cruz, California, USA), ARAF (1:1000, CST, Boston, USA), RPS6KA1 (1:500, Proteintech, Chicago, USA), GAPDH (1:30000, Proteintech, Chicago, USA), P53 (1:3000, Proteintech, Chicago, USA), p‐P53 (1:2000, Proteintech, Chicago, USA), STAT3 (1:1500, CST, Boston, USA), p‐STAT3 (1:500, CST, Boston, USA), c‐Jun (1:1000, Proteintech, Chicago, USA), p‐c‐Jun (1:2000, CST, Boston, USA), p70S6K (1:1000, Affinity, Cincinnati, USA), p‐p70S6K (1:1000, Affinity, Cincinnati, USA), P38α(1:2000, Abcam, Cambridge, UK), p‐P38α(1:1000, Abcam, Cambridge, UK), LYN (1:1000, CST, Boston, USA), p‐LYN (1:1000, CST, Boston, USA), PYK2 (1:2000, Abcam, Cambridge, UK), p‐PYK2 (1:1000, CST, Boston, USA), STAT1 (1:1000, Proteintech, Chicago, USA), p‐STAT1 (1:2000, Abcam, Cambridge, UK).

Techniques: Over Expression, Knockdown, Transfection, Migration

Journal: Cancer Medicine

Article Title: MCM8 promotes gastric cancer progression through RPS15A and predicts poor prognosis

doi: 10.1002/cam4.7424

Figure Lengend Snippet: MCM8/RPS15A axis promotes P38α, LYN, and p70S6K phosphorylation in GC. (A, B) Human Phospho‐Kinase Array Kit (ARY003C) was used to detect changes in phosphorylation of key proteins after RPS15A knockdown. (C, D) WB was applied to detect RPS15A, P53, LYN, P38α, c‐Jun, STAT1, STAT3, PYK2, p70S6K and their phosphorylated forms. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: MCM8 (1:500, Proteintech, Chicago, USA), RPS15A (1:2000, Proteintech, Chicago, USA), CDK4 (1:1000, Proteintech, Chicago, USA), MAPK14 (1:2000, Proteintech, Chicago, USA), CCND1 (1:750, CST, Boston, USA), SMAD3 (1:1000, Abcam, Cambridge, UK), SMAD4 (1:500, Santa Cruz, California, USA), ARAF (1:1000, CST, Boston, USA), RPS6KA1 (1:500, Proteintech, Chicago, USA), GAPDH (1:30000, Proteintech, Chicago, USA), P53 (1:3000, Proteintech, Chicago, USA), p‐P53 (1:2000, Proteintech, Chicago, USA), STAT3 (1:1500, CST, Boston, USA), p‐STAT3 (1:500, CST, Boston, USA), c‐Jun (1:1000, Proteintech, Chicago, USA), p‐c‐Jun (1:2000, CST, Boston, USA), p70S6K (1:1000, Affinity, Cincinnati, USA), p‐p70S6K (1:1000, Affinity, Cincinnati, USA), P38α(1:2000, Abcam, Cambridge, UK), p‐P38α(1:1000, Abcam, Cambridge, UK), LYN (1:1000, CST, Boston, USA), p‐LYN (1:1000, CST, Boston, USA), PYK2 (1:2000, Abcam, Cambridge, UK), p‐PYK2 (1:1000, CST, Boston, USA), STAT1 (1:1000, Proteintech, Chicago, USA), p‐STAT1 (1:2000, Abcam, Cambridge, UK).

Techniques: Phospho-proteomics, Knockdown

Journal: Cancer Medicine

Article Title: MCM8 promotes gastric cancer progression through RPS15A and predicts poor prognosis

doi: 10.1002/cam4.7424

Figure Lengend Snippet: Knockdown of MCM8 attenuates GC growth in vivo. (A) The left picture is the subcutaneous tumor mice. The right is the mice with the tumor removed. (B) Tumor volume and (C) weight in sh‐MCM8 and sh‐Ctrl groups. (D, E) The fluorescence images displayed the tumor burden. (F) Ki67 and (G) HE staining were detected in tissue sections. Scale bars: 100 μm. (H) IHC staining of MCM8, RPS15A, P38α, p‐38α, LYN, p‐LYN, p70S6K, and p‐p70S6K in sh‐MCM8 and sh‐Ctrl tissue sections. Scale bars: 100 μm. * p < 0.05, and *** p < 0.001.

Article Snippet: MCM8 (1:500, Proteintech, Chicago, USA), RPS15A (1:2000, Proteintech, Chicago, USA), CDK4 (1:1000, Proteintech, Chicago, USA), MAPK14 (1:2000, Proteintech, Chicago, USA), CCND1 (1:750, CST, Boston, USA), SMAD3 (1:1000, Abcam, Cambridge, UK), SMAD4 (1:500, Santa Cruz, California, USA), ARAF (1:1000, CST, Boston, USA), RPS6KA1 (1:500, Proteintech, Chicago, USA), GAPDH (1:30000, Proteintech, Chicago, USA), P53 (1:3000, Proteintech, Chicago, USA), p‐P53 (1:2000, Proteintech, Chicago, USA), STAT3 (1:1500, CST, Boston, USA), p‐STAT3 (1:500, CST, Boston, USA), c‐Jun (1:1000, Proteintech, Chicago, USA), p‐c‐Jun (1:2000, CST, Boston, USA), p70S6K (1:1000, Affinity, Cincinnati, USA), p‐p70S6K (1:1000, Affinity, Cincinnati, USA), P38α(1:2000, Abcam, Cambridge, UK), p‐P38α(1:1000, Abcam, Cambridge, UK), LYN (1:1000, CST, Boston, USA), p‐LYN (1:1000, CST, Boston, USA), PYK2 (1:2000, Abcam, Cambridge, UK), p‐PYK2 (1:1000, CST, Boston, USA), STAT1 (1:1000, Proteintech, Chicago, USA), p‐STAT1 (1:2000, Abcam, Cambridge, UK).

Techniques: Knockdown, In Vivo, Fluorescence, Staining, Immunohistochemistry

Journal: FEBS Open Bio

Article Title: Characterization of ribosome heterogeneity during endothelial to hematopoietic transition

doi: 10.1002/2211-5463.70078

Figure Lengend Snippet: The expression pattern of ribosomal proteins during EHT at the proteome level. (A) The distribution and quantity of RPL, RPS and RAP proteins were analyzed using bulk‐cell proteomic data during EHT (EC: endothelial cell, n = 3; HEC: hemogenic endothelial cell, n = 3; HC: hematopoietic cell, n = 3). (B) The average expression level of RPL, RPS and RAP proteins during EHT. (C) The stage‐specific ribosome‐related proteins during EHT. The relative fold change of average protein expression level was represented between adjacent populations. (D, E) The differentially expressed pattern of RPS15a, RPL27 and RACK1 at transcriptome (D) and proteome (E) levels.

Article Snippet: The primary antibodies were as follows: RACK1 (Proteintech, Rosemont, IL, USA; dilution 1 : 200), RPL27 (Proteintech; dilution 1 : 50), RPS15a (Boster, Pleasanton, CA, USA; dilution 1 : 50) and RPS6 (Proteintech; dilution 1 : 50).

Techniques: Expressing

Journal: FEBS Open Bio

Article Title: Characterization of ribosome heterogeneity during endothelial to hematopoietic transition

doi: 10.1002/2211-5463.70078

Figure Lengend Snippet: Quantitative analysis of ribosomal protein expression by immunofluorescence. (A) Representative immunofluorescence images showing subcellular localization of RACK1, RPL27, RPS15a and RPS6 in AECs (CD41 − CD43 − CD45 − CD31 + CD201 − Kit − CD44 + ), HECs (CD41 − CD43 − CD45 − CD31 + CD201 + Kit + CD44 + ) and HCs (CD45 + Kit + ). Scale bars = 10 μm. (B) Quantitative analysis of ribosomal protein expression through immunofluorescence signal intensity measurement. Data were collected from single AEC ( n = 6), HEC ( n = 6) and HC ( n = 5). Data are presented as the mean ± SD and analyzed by unpaired two‐tailed Student's t ‐test. Data were collected from three independent experiments.

Article Snippet: The primary antibodies were as follows: RACK1 (Proteintech, Rosemont, IL, USA; dilution 1 : 200), RPL27 (Proteintech; dilution 1 : 50), RPS15a (Boster, Pleasanton, CA, USA; dilution 1 : 50) and RPS6 (Proteintech; dilution 1 : 50).

Techniques: Expressing, Immunofluorescence, Two Tailed Test